Method for Enhancing Red Blood Cell Quality and Survival During Storage

ABSTRACT

The present invention is a method for enhancing the quality and survival of red blood cells during storage by depleting the red blood cells of both carbon dioxide and oxygen and maintaining 2,3-diphosphoglycerate acid levels.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority based on U.S. Provisional Application No. 61/376,899, filed Aug. 25, 2010, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a method for enhancing red blood cell quality and survival during storage.

Background of the Art

Anaerobic storage of red blood cells has been shown to enhance the metabolic status of red blood cells (RBC) and an increase in the potential storage time can be achieved using a variety of additive solutions. When combining an alkaline additive solution with anaerobic storage, it was observed that storage under anaerobic conditions yields insignificant benefits in terms of ATP levels. However, when the RBC additive pH was lowered from 8.1 to 6.5, significant improvement in metabolic parameters were observed under anaerobic conditions. While it has been suggested that overalkalinization of RBCs in alkaline additive solution results in increased intracellular pH due to CO₂ removed during oxygen depletion, the direct effects of CO₂ depletion on RBC quality and storage have not been demonstrated.

SUMMARY OF THE INVENTION

The present invention is a method for enhancing red blood cell quality and survival during storage by depleting a red blood cell sample of both oxygen and carbon dioxide; and transferring the oxygen- and carbon dioxide-depleted red blood cell sample to an oxygen- and carbon dioxide-impermeable environment for storage. In one embodiment, the red blood cell sample includes acidified additive solution so that 2,3-diphosphoglycerate acid levels are maintained. In some embodiments, the red blood cell sample is stored for at least three weeks and the red blood cells exhibit less than 0.2% hemolysis. In other embodiments, the red blood cells sample is stored for at least seven weeks and the red blood cells exhibit less than 0.3% hemolysis. In yet other embodiments, the red blood cells sample is stored for at least nine weeks and the red blood cells exhibit less than 0.7% hemolysis. The present disclosure provides for a method for enhancing red blood cell quality and survival during storage including the steps of reducing oxygen and carbon dioxide in a red blood cell sample; and storing the oxygen and carbon dioxide reduced red blood cell sample in an oxygen and carbon dioxide-impermeable storage environment. Adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) levels are optimized during storage in the storage environment. The present disclosure provides for a method for enhancing red blood cell quality and survival during storage including the steps of reducing oxygen and carbon dioxide in a red blood cell sample; and storing the oxygen and carbon dioxide reduced red blood cell sample in an oxygen and carbon dioxide-impermeable storage environment. Adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) levels are optimized during storage in the oxygen and carbon dioxide-impermeable storage environment.

DETAILED DESCRIPTION OF THE INVENTION

It has now been demonstrated that removal of CO₂ from RBC provides a metabolic advantage for the RBC by providing for an improved maintenance of 2,3-diphosphoglycerate acid (DPG) in the RBC. Prior to the present invention, it was shown that RBCs in an oxygen depleted environment (i.e., anaerobic) have better in vivo recovery kinetics in humans, better maintenance of ATP, and better maintenance of 2,3,-DPG) However, it was suggested that the maintenance of ATP levels in the oxygen depleted RBC could be explained not because of O₂ removal, but rather because, under the experimental conditions, there was a concomitant removal of CO₂ leading to alkalization (i.e., increase in pH) of the RBC that in turn has a direct effect on the enzyme phosphofructokinase, a rate limiting factor in glycolysis. It has been suggested that with an acidified additive solution under anaerobic conditions, an increase in ATP is due primarily not from the pH effect on glycolysis, but rather through the effect of the deoxygenated hemoglobin binding free 2,3-DPG, wherein alkalinization may play a secondary role in ATP maintenance in the anaerobically stored RBC. Experiments where carbon monoxide was used to displace 2,3,-DPG from the deoxygenated hemoglobin were shown to provide no improvement in ATP levels over storage (i.e., ATP was similar to oxygen stored RBC).

Based upon the above suggestions, one would expect that CO₂ depletion would contribute to alkalization of the RBC cytosol in addition to the alkalization effect of protons (H+) being bound by deoxygenated hemoglobin. This additional alkalization from CO₂ depletion would then increase the flux through glycolysis (Scheme 1) because of the effect on the phosphofructokinase enzyme and increase the production of ATP.

As described in Table 1, maintaining CO₂ when O₂ was depleted matched the pH of the control aerobic condition whereas CO₂ removal resulted in alkalinization. ATP was higher in the CO₂ replete anaerobic arm than the control and the CO₂ depleted anaerobic arm. Glycolysis rate, as indicated by lactate accumulation and glucose consumption, was equivalent in the CO₂ replete arm than in the control arm. Glycolysis rate was slightly greater in the CO₂ depleted anaerobic arm that the other two, but ATP in this arm was lower than the CO₂ replete arm that has a pH the same as the control arm. These observations indicate that the main ATP metabolic advantage for anaerobic storage is not through the pH affect on phosphofructokinase. Further, ATP association with hemoglobin and less ATP utilization through the pentose phosphate pathway or other pathways may be important contributors to the maintenance of ATP over storage in the anaerobic environment. However, unexpectedly, 2,3-DPG was depleted in the CO₂ replete anaerobic arm like the control, indicating that there is a previously undescribed effect of CO₂ (perhaps through pH) on diphosphoglycerate mutase and/or diphosphoglycerate phosphatase in DPG synthesis or other pathway.

TABLE 1 Anaerobic Control with CO₂ Anaerobic with Results Aerobic preservation¹ CO₂ depletion² Expected Glycolysis Control Equal to Greater than (Lactate) control control ATP Control Greater than Greater than OK control control (because Perhaps anaerobic) slightly greater than CO₂ replete 2,3-DPG Control Greater than Greater than depleted control control (because (through anaerobic) ~21 days) Observed Glycolysis Control Equal to Greater than (Lactate) control control Control Greater than Greater than OK control control Greater Less than CO₂ than CO₂ replete** depleted** Observed 2,3-DPG Control Equal to Greater than depleted control** control Less (through than CO₂ ~21 days) depleted** Greater than CO₂ replete** ¹Ar/CO₂ purged and Ar/CO₂ stored. ²Ar purged and Ar stored. **Unexpected results.

DPG was not maintained by the association with O₂-depleted hemoglobin as shown in the CO₂ replete anaerobic arm. DPG was maintained in the CO₂ depleted arm. After approximately day 21 DPG did fall along with pH. This DPG fall may be associated with pH, indicating that enzymes in the DPG synthesis pathway may be affected by pH. Therefore, to maintain DPG in anaerobic storage, removal of CO₂ before and during storage of RBC is required.

Accordingly, the present invention is a method for enhancing red blood cell quality and survival during storage by depleting a red blood cell sample of both oxygen and carbon dioxide; and transferring the oxygen- and carbon dioxide-depleted red blood cell sample to an oxygen- and carbon dioxide impermeable environment. For the purposes of this invention, a red blood cell sample refers to whole blood; anti-coagulated whole blood (AWB); packed red cells obtained from AWB; and red cells separated from plasma and resuspended in physiological fluid. A red blood cell sample is typically supplied in a source container and can include any treated or untreated fluid from a living organism that contains red blood cells, particularly blood, including whole blood, warm or cold blood, and fresh blood; treated blood, such as blood diluted with a physiological solution, including but not limited to saline, nutrient, and/or anticoagulant solutions; analogous blood products derived from blood or a blood component. The red blood cell sample may include leukocytes, may be treated to remove leukocytes, may be treated with gamma or X-ray irradiation, washed, or treated to reduce or eliminate pathogens.

Depletion of both oxygen and carbon dioxide from the red blood cell sample can be achieved using any technique or combination of techniques described herein. For example, the instant method can employ gas purging and/or selective removal of O₂ and/or CO₂ with, e.g., a gas permeable membrane, an O₂ and/or CO₂ adsorbent, a molecular imprinted polymer, or a combination thereof. Techniques for purging blood via gas exchange with an inert gas such as argon are well-known and routinely practiced in the art. Gas permeable membranes have also been developed for removing O₂ and/or CO₂ from a liquid. Typically, the membranes are formed into hollow fibers and packaged in membrane modules, wherein the rate of gas transfer across the membrane is proportional to the gas permeability coefficient, the membrane surface area, the trans-membrane gas partial pressure difference, and inversely proportional to the membrane thickness. Exemplary gas permeable membrane modules of use in depleting oxygen and/or carbon dioxide are available from commercial sources. For example, PermSelect® Silicone Hollow Fiber Membranes, available from MedArray Inc. (Ann Arbor, Mich.); and Liqui-Cel® Membrane Contactors, available from Membrana-Charlotte (Charlotte, N.C.), are marketed for use in depleting oxygen and carbon dioxide from liquids in pharmaceutical and medical applications.

“Adsorbent” for the present purposes refers to a porous solid, particulate material or mixture of materials, which selectively admits and retains within its pores (or adsorbs) O₂ and/or CO₂ from a liquid. Suitable adsorbents for use in the present method are those having good selectivity for O₂ and/or CO₂ over other constituents (e.g., N₂), good kinetics, high durability, good chemical compatibility, and reasonably low cost. For example, molecular sieves are materials whose atoms are arranged in a lattice or framework in such a way that a large number of interconnected uniformly sized pores exist. The pores generally only admit molecules of a size about equal to or smaller than that of the pores. Molecular sieves, thus, can be used to adsorb and separate or screen molecules based on their size with respect to the pores. One class of molecular sieves is zeolites, which have been shown to exhibit exceptional selective capture and storage of CO₂. Zeolites are hydrated silicates of aluminum. As such, zeolites, on account of their chemical composition, are part of a broader class of adsorbents called aluminosilicates. Other molecular sieves are formed from aluminophosphates, called ALPO₄ ¹S, titanosilicates, metalloaluminates, etc. Zeolites can be naturally occurring or artificial. Activated alumina, activated carbon, and silica gel are other broad classes of adsorbents that could be used to capture CO₂. In some embodiments, the adsorbents are attached to a substrate (e.g., a bead, pellet, granule or particle) to facilitate contact with and removal of the adsorbents from the RBC.

A molecular imprinted polymer (MIP) is a polymer formed in the presence of a molecule that is extracted afterwards, thus leaving complementary cavities behind. These polymers show a chemical affinity for the original molecule and are of use in sensing and separation methods. For example, metal complexing imprinted polymers have been prepared for gas molecules such as NO, CO, CO₂ and oxygen, wherein the imprinted cavities in the polymer matrices were sized to the appropriate gas molecules used as the template. Moreover, copolymerization of these metal complexes into organic hosts such as porous methacrylate polymers has been shown to provide a substrate for binding gaseous molecules such as CO. Accordingly, molecular imprinted polymers in a bead, pellet, granule or particle format can be used in removal of CO₂ and oxygen in the instant method.

As exemplified herein, gas purging can achieve a pCO₂ of about 5 mmHg and a pO₂ of about 10 mmHg. Accordingly, in particular embodiments, the oxygen- and carbon dioxide-depleted red blood cell sample of the invention has a pCO₂ of less than or equal to about 5 mmHg and a pO₂ of less than or equal to about 10 mmHg. Alternatively, in so far as gas permeable membranes can deplete the oxygen in a liquid to a level of at least 1 ppb and CO₂ to a level of at least 1 ppm, other embodiments of this invention include depleting oxygen and carbon dioxide in the red blood cell sample to at least 1 ppb and 1 ppm, respectively. As is routine in the art, a pO₂ needle probe, or pO₂ and pCO₂ microelectrode can be used to measure oxygen and carbon dioxide levels in the oxygen- and carbon dioxide-depleted red blood cell sample.

Once the red blood cell sample is depleted of both oxygen and carbon, the red blood cell sample is transferred to an oxygen- and carbon dioxide-impermeable environment for storage. As used herein, an oxygen- and carbon dioxide-impermeable environment is a storage container or storage container system that is impermeable to oxygen and carbon dioxide. In accordance with the present invention, an oxygen- and carbon dioxide-impermeable storage container is a container, pouch, bag, or bottle that is constructed of a material compatible with a biological fluid, such as whole blood or a blood component and is capable of withstanding centrifugation and sterilization. Such containers are known in the art and include, e.g., for example, blood collection and satellite bags. Storage containers of use in the instant method can be made of plasticized polyvinyl chloride, e.g., PVC plasticized with dioctylphthalate, diethylhexylphthalate, or trioctyltrimellitate. The bags may also be formed from polyolefin, polyurethane, polyester, and polycarbonate. In one embodiment, the storage container itself is constructed of an oxygen- and carbon dioxide-impermeable material. Impermeable materials are routinely used in the art and any suitable material can be used. Existing systems use oxygen- and carbon dioxide-impermeable receptacles composed of layers of ethylene vinyl alcohol copolymer and modified ethylene vinyl acetate copolymer, impermeable to oxygen and carbon dioxide ingress. In another embodiment, the storage container is a component of a storage container system that is impermeable to oxygen and carbon dioxide. Such systems include, but are not limited to, use of an oxygen- and carbon dioxide-impermeable over wrap or over bag which encloses the storage container.

To ensure that there is no ingress of oxygen or carbon dioxide into the sample during transfer, in particular embodiments, the oxygen- and carbon dioxide-depleted red blood cell sample is transferred to the storage container under positive pressure. Positive pressure is a pressure within a system that is greater than the environment that surrounds that system. Positive pressure can be attained by transferring the sample to the storage container in a closed system, e.g., via tubing between one or more of the source container, oxygen and carbon dioxide depletion apparatus, and storage container. Airtight, pressurized fluid delivery systems for facilitating positive fluid flow are known in the art. Transfer of the oxygen- and carbon dioxide-depleted red blood cell sample to the storage container can be achieved using various techniques including, but not limited to peristalsis, a siphon, or a combination thereof. By way of illustration, the red blood cell sample can be transferred via a tube from the source container to a gas permeable membrane module or molecular sieve device and subsequently to the source container via another tube, wherein the source container, membrane or sieve, and storage container are positioned in an inverted siphon configuration.

As used herein, tubing can be any conduit or means which provides fluid communication between containers, and is typically made from the same flexible material as is used for the containers, and is desirably oxygen- and carbon dioxide impermeable. The tubing may extend into the interior of the containers herein, and may be used as a siphon, for example. There may be a number of tubes providing fluid communication to any individual container, and the tubes may be oriented in a number of ways. A seal, valve, clamp, transfer leg closure, or the like can also be located in or on the tubing. It is intended that the present invention is not limited by the type of material used to construct the containers or the conduit which connects the containers.

Once transferred to the storage container, the red blood cell sample can be stored under aerobic or anaerobic conditions, i.e., conditions of low or no oxygen. Desirably the sample is stored between 1° C. and 6° C. to further enhance the survival of the red blood cells.

Compared to a red blood cell sample not prepared in accordance with the present method, the quality and survival of the red blood cell sample of the invention is enhanced. In this respect, the red blood cells of the instant sample exhibit less than 0.7%, hemolysis at 9 weeks, less than 0.3% hemolysis at 7 weeks, and less than 0.2% hemolysis at 3 weeks after treatment by the instant method. The determination of red blood cell hemolysis is routinely practiced in the art and any suitable method can be employed.

Moreover, when the instant method is carried out in the presence of acidified additive solution (i.e., an additive solution of between pH 5.5 and 7.0, or more desirably between 6.25 and 6.75), 2,3-DPG levels of the RBCs are maintained at higher levels than controls (e.g., a sample wherein carbon dioxide is not depleted); i.e., 2,3-DPG levels are approximately 60% higher than controls at 3 weeks. 2,3-DPG levels are routinely measured and any suitable method can be used to determined whether 2,3-DPG levels of a red blood cell sample produced by the instant method are being maintained above control samples.

By maintaining 2,3-DPG levels within the RBC, the RBC can provide better oxygen transfer to tissues when the RBC is transfused to patients. This improvement can also provide improved recovery of transfused RBC in patients and study subjects. The present finding provides a mechanism to increase the acceptable storage time of RBC for transfusion or other purposes. In this respect, the red blood cells of the red blood cell sample can be used, e.g., in a transfusion, for at least 4, 5, 6, 7, 8, or 9 weeks after treatment by the instant method.

Example 1: Materials and Methods

It was determined whether removal of CO₂ during oxygen depletion by gas exchange affects RBC in a significant manner during subsequent anaerobic storage. Using a three-way, split-unit study, 12 units (6 units with AS3 and 6 with OFAS3) were evaluated, which compared anaerobically stored RBC units with oxygen depletion accomplished using 100% Ar vs. a 95% Ar/5% CO₂ gas mixture. As a control, one of the three-way split units was stored conventionally in AS3 (also known as NUTRICEL, Pall Corp.)(Hess, et al. (2000) supra) additive or the OFAS3 (Dumont, et al. (2009) supra).

The primary endpoints of this study included the weekly determination of biochemical parameters such as hemoglobin, extracellular pH (pHe), internal pH (pHi), partial pressure of carbon dioxide (pCO₂), partial pressure of oxygen (pO₂), hematocrit, ATP, 2,3-DPG levels, supernatant hemoglobin, glucose, lactate, Na+, K+ and % hemolysis carried out during nine weeks of refrigerated storage.

Preparation of Red Cells for Storage.

Each unit of blood (500 mL) was collected into LEUKOTRAP RC Whole Blood Collection, Filtration and Storage System with Pall RC2D Filter containing CP2D in the primary bag. The blood was held at room temperature (RT) for 30 minutes, prior to centrifugation. Subsequently, the blood was centrifuged at 2,000×g for 3 minutes (‘soft spin’-slow stop and no brake), and the supernatant (platelet-rich plasma fraction) was expressed into the attached satellite bag and discarded. After centrifugation, additive solution (100 mL of AS-3 or 200 mL of OFAS3) was added to the unit of packed red blood cells (pRBC). When OFA was being added, the pre-prepared bag was sterilely docked to the pRBC collection bag set. The pRBC unit was mixed very well with the “blood vortex” method-opposite end rotation. Subsequently, the pRBC unit in additive solution was leukoreduced at room temperature using the attached RC2D filter. The pRBC unit was divided equally into three 600 mL bags by mass for the following treatment: Control, 100% Ar, and 5% CO₂/Ar. Before transferring the pRBC unit into the three 150 mL bags, each bag was purged with the appropriate gas: control (none), 100% Ar, or 5% CO₂/Ar. Upon transfer, the control bag was mixed for 70 minutes on an agitator at room temperature. For the 100% Ar bag and 5% CO₂/Ar bag, the RBC was depleted of oxygen as described herein. Each 150 mL bag was sampled by sterilely docking a plasma transfer set (syringe with tubing). After removal of the 9 mL of pRBC for testing, the bag was purged completely of “gas head” with a syringe. Each unit was placed within a temperature monitored blood storage refrigerator which maintained the temperature at 4° C. On a weekly basis for 9 weeks, each unit was mixed and sampled.

Oxygen Depletion with 100% Argon.

Argon was filter sterilized through a 0.22 micron hydrophilic filter and introduced into the pRBC bag. Care was taken not to pressurize the bag at this point. The bag was gently mixed with a rocking motion for 10 minutes at 21-25° C., then the gas was gently expressed through the filter using a vacuum. Flushing with Argon gas, gentle mixing, and gas phase expression was repeated six additional times at 21-25° C. (for a total of seven times). After the final exchange, a 9 mL sample was taken from the bag. Analysis of oxygen and carbon dioxide levels in the depleted RBC indicated that the pCO₂ was about 5 mmHg and the pO₂ was about 10 mmHg (at a temperature of from 21-25° C. The bags were stored in a gas-tight canister containing a Pd catalyst (DIFCO). The vacuum was set at ˜−0.7 bar (1 bar=0.987 standard atmosphere). The canister was filled with Ar to ˜0.7 bar. The canister was evacuated to ˜−0.7 bar, and filled with 10% H₂/90% Ar gas mixture to +0.3 bar. Hydrogen and the Pd catalyst form a fully functional oxygen scavenging system. The canister was placed within a temperature monitored blood storage refrigerator, which maintained 4° C.

Oxygen Depletion with 95% Argon/5% CO₂.

A gas mixture of Argon/5% CO₂ gas was filter sterilized through a 0.22 micron hydrophilic filter and introduced into the bag. Care was taken not to pressurize the bag at this point. The bag was gently mixed with a rocking motion for 10 minutes at 21-25° C., and the gas was gently expressed through the filter using a vacuum. Flushing with the gas mixture, gentle mixing, and gas phase expression, were repeated six additional times at 21-25° C. (for a total of seven times). After the final exchange, a 9 mL sample was taken from the bag. The bags were stored in a gas-tight canister containing a Pd catalyst. The vacuum was set at ˜−0.7 bar. The canister was filled with Ar to ˜0.7 bar. The canister was evacuated to ˜−0.7 bar, and filled with 5% C0₂/10% H₂/90% Ar gas mixture to +0.3 bar. The canister was placed within a temperature monitored blood storage refrigerator, which maintained 4° C.

Example 2: CO₂ Depletion Provides a Metabolic Advantage for Stored RBC

The instant analysis was a matched three arm study including a control sample, a sample depleted of O₂ and CO₂ with Ar, and a sample depleted of O₂ with 95% Ar/5% CO₂. Whole blood was collected into CP2D (Pall), centrifuged 2000×g for 3 minutes, plasma removed, and additive solution AS-3 (Nutricel, Pall), or experimental OFAS3 added. The unit was evenly divided into three 600 mL bags. Two bags were gas exchanged with Ar or Ar/CO₂, transferred to 150 mL PVC bags and stored at 1-6° C. in anaerobic cylinders with Ar/H₂ or Ar/H₂/CO₂. One control bag was treated in the same manner without a gas exchange and stored at 1-6° C. in ambient air. Bags were sampled weekly for up to 9 weeks and a panel of in vitro tests were conducted on each sample including intra- and extra-cellular pH (pHi, pHe).

As shown in Table 2, purging with Ar resulted in alkalization of the RBC and upregulation of glycolysis compared to control. pH and lactate of Ar/CO₂-purged RBC were equivalent to aerobically stored controls (p>0.5, days 0-21). ATP levels were higher in Ar/CO₂ (p<0.0001). DPG was maintained beyond 2 weeks in the Ar-purged arm only (p<0.0001). Surprisingly, DPG was lost at the same rate in both control and Ar/CO₂ arms (p=0.6). Hemolysis was low in all arms, but may have been influenced by the weekly mixing.

By reducing carbon dioxide and oxygen in the red blood cell ATP levels were maintained at higher levels for nine weeks relative to ATP levels in a red blood cell sample in which neither oxygen nor carbon dioxide were depleted. 2,3-DPG levels were maintained at a higher level for three weeks than 2,3-DPG levels in a red blood cell sample in which neither oxygen nor carbon dioxide were depleted. Oxygen depletion has a positive impact on ATP levels in red blood cell samples and carbon dioxide depletion has a positive impact on 2,3-DGP levels. Optimal results are achieved when both oxygen and carbon dioxide are depleted.

Although the present disclosure describes in detail certain embodiments, it is understood that variations and modifications exist known to those skilled in the art that are within the disclosure. Accordingly, the present disclosure is intended to encompass all such alternatives, modifications and variations that are within the scope of the disclosure as set forth in the disclosure.

TABLE 2 ATP 2,3-DPG Lactate Glucose Hemo- pHi (μmol/ (μmol/ (mmol/ (mmol/ lysis Arm Day (22° C.) gHb) gHb) gHb) gHb) (%) Control 0 7.11 ± 4.1 ± 12.2 ± 0.01 ± 0.38 ± 0.20 ± 0.04 0.7 1.8 0 0.13 0.04 21 6.77 ± 4.3 ± 0.2 ± 0.11 ± 0.30 ± 0.20 ± 0.05 1.0 0.2 0.02 0.13 0.06 42 6.56 ± 3.1 ± 0.2 ± 0.16 ± 0.27 ± 0.30 ± 0.06 0.8 0.1 0.02 0.13 0.10 63 6.44 ± 2.1 ± 0.3 ± 0.2 ± 0.26 ± 0.53 ± 0.07 0.6 0.2 0.02 0.13 0.20 Ar/CO₂ 0 7.14 ± 4.6 ± 12.2 ± 0.02 ± 0.38 ± 0.17 ± 0.03 0.7 1.8 0 0.12 0.04 21 6.76 ± 5.5 ± 0.2 ± 0.1 ± 0.33 ± 0.19 ± 0.04 1.3 0.2 0.02 0.13 0.08 42 6.58 ± 4.0 ± 0.1 ± 0.15 ± 0.30 ± 0.32 ± 0.05 1.3 0.0 0.03 0.13 0.11 63 6.51 ± 2.4 ± 0.1 ± 0.19 ± 0.28 ± 0.61 ± 0.06 1.0 0.0 0.03 0.13 0.26 Ar 0 7.38 ± 4.6 ± 14.3 ± 0.02 ± 0.38 ± 0.18 ± 0.06 0.8 1.6 0 0.13 0.05 21 6.67 ± 4.7 ± 6.2 ± 0.16 ± 0.33 ± 0.19 ± 0.04 0.9 3.0 0.02 0.14 0.04 42 6.42 ± 3.3 ± 0.4 ± 0.21 ± 0.32 ± 0.28 ± 0.06 0.9 0.2 0.02 0.13 0.08 63 6.31 ± 1.8 ± 0.4 ± 0.24 ± 0.3 ± 0.64 ± 0.09 0.9 0.2 0.02 0.14 0.35 mean ± sd

The results of this analysis indicated that the addition of 5% CO₂ to the purging gas prevented CO₂ loss with an equivalent starting pHi and pHe to control bags. Maintenance of ATP in the Ar/CO₂ arm demonstrated that ATP production was not solely a function of the pH effect on glycolysis. CO₂ in anaerobic storage prevented the maintenance of DPG, and DPG appeared to be pH dependent. Therefore, CO₂ as well as O₂ depletion provided metabolic advantage for stored RBC. 

1.-18. (canceled)
 19. A stored oxygen and carbon dioxide depleted red blood cell composition comprising: oxygen and carbon dioxide depleted packed red blood cells; reduced leukocytes; reduced platelets; reduced pathogens; and an acidified additive solution.
 20. The composition of claim 19, wherein said oxygen and carbon dioxide depleted packed red blood cells comprise an oxygen level of 10 mmHg or less.
 21. The composition of claim 19, wherein said oxygen and carbon dioxide depleted packed red blood cells comprise a carbon dioxide level of 5 mmHg or less.
 22. The composition of claim 19, wherein said composition exhibits less than 0.2% hemolysis after three weeks of storage at 1 to 6° C.
 23. The composition of claim 19, wherein said composition exhibits less than 0.3% hemolysis after three weeks of storage at 1 to 6° C.
 24. The composition of claim 19, wherein said composition exhibits increased 2,3-DPG level for up to 42 days of storage compared to non-depleted red blood cells under the same storage conditions.
 25. The composition of claim 19, wherein said composition exhibits increased 2,3-DPG level for at least three weeks of storage compared to non-depleted red blood cells under the same storage conditions.
 26. The cell composition of claim 19, wherein said composition maintains 2,3-DPG level for at least two weeks.
 27. The red blood cell composition of claim 19, wherein said composition maintains 2,3-DPG level for at least three weeks compared to control.
 28. The composition of claim 19, wherein said composition maintains ATP levels at a higher level compared to control.
 29. The composition of claim 19, wherein said acidified additive solution has a pH ranging from 5.5 to 7.0.
 30. The composition of claim 19, wherein said acidified additive solution has a pH between 6.25 and 6.75.
 31. The composition of claim 19, wherein said stored oxygen and carbon dioxide depleted red blood cell composition is stored for at least three weeks.
 32. The composition of claim 19, wherein said stored oxygen and carbon dioxide depleted red blood cell composition is stored for at least seven weeks.
 33. The composition of claim 19, wherein said red blood cell composition further comprises saline.
 34. The composition of claim 19, wherein said red blood cell composition further comprises nutrients.
 35. The composition of claim 19, wherein said red blood cell composition further comprises an anticoagulant solution. 